Research Service, Miami VAHS, Miami, Florida, USA.
Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida, USA.
Exp Lung Res. 2023;49(1):152-164. doi: 10.1080/01902148.2023.2246074.
Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells' gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Incubation of iAT2 cells with LPS increased expression of IL1-β and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-β, TNF-α, TGFβ-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.
生长激素释放激素（GHRH）是一种 44 个氨基酸的肽，可调节生长激素（GH）的分泌。我们假设肺泡Ⅱ型（AT2）细胞中的 GHRH 受体（GHRH-R）可以调节脂多糖（LPS）或细胞因子的促炎和可能随后的促纤维化作用，使得 AT2 细胞可以参与肺炎症和纤维化。我们使用人诱导多能干细胞（iPSC）衍生的肺泡Ⅱ型（iAT2）上皮细胞来研究 GHRH-R 如何调节基因和蛋白质表达。我们测试了 iAT2 细胞对 LPS 或细胞因子的基因表达反应，以寻找这些机制是否导致促炎分子或间充质标志物的内源性产生。实时定量 PCR（RT-PCR）和 Western blot 用于研究上皮和间充质标志物的差异表达。LPS 孵育增加了 iAT2 细胞中 IL1-β 和 TNF-α 以及间充质基因（包括 ACTA2、FN1 和 COL1A1）的表达。GHRH-R 肽拮抗剂 MIA-602 显著抑制了 LPS 引起的肺泡上皮细胞基因表达。与纤维化肺中存在的细胞因子类似，孵育 iAT2 细胞会增加 IL1-β、TNF-α、TGFβ-1、Wnt5a、平滑肌肌动蛋白、纤维连接蛋白和胶原蛋白等基因的表达。间充质蛋白（如 N-钙粘蛋白和波形蛋白）的表达在长时间暴露于细胞因子后也升高，证实了上皮细胞产生促炎分子是导致随后纤维化的重要机制。iAT2 细胞显然表达 GHRH-R。LPS 或细胞因子暴露增加了 iAT2 细胞促炎因子的产生。GHRH-R 拮抗剂 MIA-602 抑制了促炎基因表达，表明 iAT2 细胞 GHRH-R 信号参与了肺炎症，并且可能参与了纤维化。
